Master’s Thesis 2016
Department of Chemistry, Biotechnology and Food Science
How does a low-FODMAP diet affect the gut microbiota composition in patients with irritable bowel
syndrome?
Hvordan påvirkes tarmflorasammensetningen til pasienter med irritabelt tarmsyndrom av en lav-FODMAP diett?
Silje Søyland Amundsen
Food science;; Food, Health and Nutrition
ACKNOWLEDGEMENTS
I would like to thank my primary supervisor Professor Harald Carlsen at Norwegian University of Life Sciences, Campus Ås and my co-supervisor Anne Marie Aas at the institute of clinical medicine, University in Oslo. Harald and Anne have always helped me when I have stagnated and they have given me good guidance during this semester. Thanks for their help, I have learned a lot in a short time.
I would also like to thank all my dearest friends, and my family, particularly Silje K. Hansen (#Siljepower) for proofreading, for supportive words during this semester and for being my bestfriend. Additionally, I would like to thank Jon Ola Olausson, Gina Marie Qvale, Marit K.
Strand, and Susanne B. Sørensen as well as my dad, my mom and my siblings. They have motivated me as well as supported me when I needed it the most. This is in addition to remind me that life are of more than writing a master thesis. They are morally complicit in that I have produced a thesis I'm proud of, and I´m forever grateful!
I would also like to thank Lotte-Martine Lagerholt for being my un-biological twin-sister, and for all she has had to accept during this semester. Finally, I would like to thank all my fellows at the reading hall at Sørhellinga, Campus Ås, for all the fun times and for making the best out of each situation.
As my dad says “Always do your best, but you´re doing that already”
Ås, May 2016
Silje S. Amundsen
ABSTRACT
Background: Irritable bowel syndrome (IBS) is a disorder characterized by abdominal pain, diarrhea, constipation and general discomfort. It is also characterized by a change in the composition of microorganisms in the intestine, which is referred to as gut microbiota dysbiosis. This disorder is regarded as a multifactorial disorder, although the pathophysiology remains controversial. Dietary strategies have been employed to reduce symptoms of IBS. However, diets, particularly the low- FODMAP diet, which appears to reduce symptoms, may not be optimal with respect to a healthy gut microbiota composition.
Aim: To determine to which extent dietary strategies used against IBS, particularly the low-FODMAP diet, alter gut microbiota in IBS patients, and to further discuss whether such changes are beneficial or not
Methods: A literature search was conducted using various terms, some examples; ´irritable bowel syndrome or IBS´, ´nutrition or diet´, ´RCT or randomized controlled trial or epidemiology or pilot´
Results: A diet low in fermentable carbohydrates change the gut microbiota composition, however whether this is beneficial or not to IBS patients is difficult to determine due to different findings and the short duration of these studies. Despite this, there is evidence to state that the gut microbiota has changed. This includes a probable switch from carbohydrate to protein metabolism by bacteria belonging to the genera Bacteorides, Porphyromonas, Clostridium and Adlercreutzia, despite not confirmed by changes in BCFAs or protein metabolites. In addition, it is a decrease of the lactate- producing probiotic bacteria, and of the mucus-associated A. muciniphila. Furthermore, there are increased levels of the mucus-associated bacteria R. torques as well as both a decrease and an increase of Roseburia spp. Finally, an increase of both gas-producing and gas-consuming bacteria were seen in non-responders, while responders were depleted for these. The degradation of proteins by the gut microbiota might have detrimental effects, due to the observed association between increased genotoxicity and protein metabolites in previous studies. However, further studies are needed, specifically on the long-term effect of carbohydrate restriction on the gut microbiota composition in IBS patients. Further research is also needed of the reintroduction phase of a fermentable carbohydrate restriction diet, to examine whether the changes in the gut microbiota composition in IBS patients following a low-FODMAP diet are being reversed or persists. Finally, further studies on the gut microbiota to non-responders in comparison to responders are needed.
Conclusion: A low-FODMAP diet alters the gut microbiota composition in patients with IBS, and it seems like this diet result in a trend toward adversely effects on the hosts´ health, despite not confirmed by changes in BCFAs. However, whether this diet is harmful for the host over time or not needs further research.
SAMMENDRAG
Bakgrunn: Irritabelt tarmsyndrom (IBS) er en lidelse karakterisert av mageknip, diaré, forstoppelse, flatulens, oppblåsthet og generell ubehag, i tillegg til en dysbiose, en endring i sammensetningen av tarmbakterier. IBS er en multifaktoriell lidelse, men årsakene er enda uklare. Forskjellige dietter har blitt foreslått for å redusere symptomene som oppstår hos IBS pasienter, spesielt en lav-FODMAP (Fermenterbare Oligo-, Di-, Monosakkarider And Polyoler) diett, men det kan tyde på at denne dietten er lite gunstig med hensyn på tarmflorasammensetningen hos IBS pasienter.
Mål: Å finne ut i hvilken grad koststrategier som reduserer symptomer hos IBS pasienter, spesielt en lav-FODMAP diett, påvirker tarmflorasammensetningen hos IBS pasienter, og ytterligere da diskutere om slike endringer er gunstige eller ikke.
Metoder: Et litteratursøk ble utført i PubMed og Cochrane, og følgende søkeord ble benyttet: ´irritable bowel syndrome or IBS´, ´nutrition or diet´, ´RCT or randomized controlled trial or epidemiology or pilot´
Resultater: I denne masteroppgaven ble det vist at en restriksjon av fermenterbare karbohydrater endrer tarmflorasammensetningen. Om dette er fordelaktig for vertens helse eller ikke, er vanskelig å bestemme, grunnet at studiene som ble inkludert i denne masteroppgaven observerte mye forskjellig. I tillegg var disse studiene korttidsstudier (1-4 uker). Uansett, en slik diett kan ha vist en trend mot et metabolismebytte hos tarmbakteriene hos IBS pasienter, fra karbohydrat- til proteinmetabolisme, til tross for at dette ikke ble støttet av målingene av BCFAs eller andre proteinmetabolitter. Spesielt av bakterier som hører til slektene Bacteroides, Porphyromonas, Clostridium og Adlercreutzia spp. Når tarmbakterier degraderer proteiner i den distale colon (tykktarm) produseres endeprodukter som har blitt assosiert med økt genotoksisitet hos mus. Videre forskning trengs, før en med sikkerhet kan si at en low- FODMAP diett gir negative effekter. Videre ga denne dietten en reduksjon av laktat-produserende probiotiske bakterier, og av den mucus-assosierte bakterien A. muciniphila. Det var og en økning av den mucus-assosierte bakterien R. torques, så vel som en reduksjon og en økning av Roseburia spp.
Gassproduserende bakterier så vel som bakterier som konsumerer gass var økt hos non-responders, mens responders hadde lite av disse bakteriene. Det trengs videre forskning, spesielt på langtidseffekten av en lav-FODMAP diett på tarmflorasammensetningen hos IBS pasienter. I tillegg trengs det studier på effektene av reintroduksjonsfasen av en lav-FODMAP diett, for å undersøke om endringene som er vist i tarmflorasammensetningen hos IBS pasienter vedvarer eller reverseres. Det trengs og mer forskning på tarmflorasammensetningen hos non-responders sammenlignet med tarmflorsammensetningen hos responders.
Konklusjon: En lav-FODMAP diet endrer tarmflorasammensetningen hos pasienter med IBS, og det kan se ut som at denne dietten heller mot negative effekter for vertens helse. Om dette er effekter som er skadelige for verten over tid eller ikke trenger videre forskning.
ABBREVIATIONS
BCFAs: Branch-Chain Fatty Acids BAM: Bile-Acid Malabsorption DCA: Deoxycholic Acid
FGID: Functional GastroIntestinal Disease
FISH: Fluorescence in situ hybdridization (abbreviation used in Tables) FODMAP: Fermentable Oligo-, Di-, Monosaccharides And Polyols FOS: Fructo-oligosaccharides
GI: Gastrointestinal
GOS: Galacto-oligosaccharides
HFM: High-FODMAP Diet (abbreviation used in Tables) IBS: Irritable Bowel Syndrome
IBS-C: Constipation predominant irritable bowel syndrome IBS-D: Diarrhea predominant irritable bowel syndrome
IBS-M: Mixed irritable bowel syndrome (diarrhea and constipation) IBS-U: Unsubtyped irritable bowel syndrome
LBHT: Lactulose Breath Hydrogen Test LFSD: Low Fermentable Substrate Diet
LFM: Low-FODMAP diet (abbreviation used in Tables) OTU(s): Operational Taxonomic Unit(s)
PCR: Polymerase Chain Reaction pHBA: p-hydroxybenzoic acid PI-IBS: Postinfectious IBS
RCT: Randomized Controlled/Clinical Trial SCFAs: Short-Chain Fatty Acids
SIBO: Small Intestinal Bacterial Overgrowth
TAD: Typical Australian Diet (abbreviation used in Tables) WGTT: Whole Gut Transit Time
TABLE OF CONTENT
ACKNOWLEDGEMENTS ... II ABSTRACT ... IV SAMMENDRAG ... VI ABBREVIATIONS ... VIII
LIST OF FIGURES ... 2
LIST OF TABLES ... 2
1 INTRODUCTION ... 3
1.1 IRRITABLE BOWEL SYNDROME ... 3
1.1.1 Definition, diagnoses and classification ... 3
1.1.2 Prevalence of IBS ... 5
1.1.3 Etiology of IBS ... 6
1.2 GUT MICROBIOTA ... 8
1.2.1 Definition and function ... 8
1.2.2 Taxonomy ... 8
1.2.3 What characterizes a healthy gut microbiota? ... 11
1.2.4 Gut microbiota and diet interaction ... 12
1.2.5 Gut microbiota and IBS ... 15
1.3 DIETARY TREATMENT AND MANAGEMENT OF IBS ... 17
1.3.1 Low-‐FODMAP diet ... 17
2 AIM ... 20
3 METHODS ... 21
3.1 Study selection ... 21
3.2 Quality of the studies using the Jadad scale ... 23
4 RESULTS ... 25
4.1 Quality of studies and compliance to dietary interventions ... 25
4.2 Effect of fermentable carbohydrate restriction on IBS symptoms, microbial metabolites and the gut microbiota composition ... 26
4.2.1 Low-‐FODMAP diet and effect on IBS symptoms ... 26
4.2.2 Gut microbial products/metabolites ... 26
4.2.3 Gut microbiota composition at baseline ... 27
4.2.4 Gut microbiota following a fermentable carbohydrate restriction diet ... 27
Summary ... 32
5 DISCUSSION ... 34
5.1 Overall quality of the included trials ... 34
5.1.1 Quality of the four included studies ... 34
5.1.2 Adherence to the diet ... 35
5.2 Effect of a fermentable carbohydrate restriction of the gut microbiota composition and microbial metabolites in IBS patients ... 36
5.2.1 Changes at the phyla level ... 36
5.2.2 Probiotic bacteria ... 36
5.2.3 Butyrate-‐producing bacteria ... 37
5.2.4 SCFA-‐production ... 38
5.2.5 Protein-‐associated bacteria ... 39
5.2.6 Mucus-‐associated bacteria ... 40
5.2.7 Gas-‐producing bacteria ... 41
6 CONCLUSION ... 42
7 REFERENCES ... 43
LIST OF FIGURES
Figure 1: Worldwide prevalence of IBS 13 ... 5Figure 2: Phylogenetic tree of some of the bacteria belonging to the phylum Firmicutes (according to NCBI). Phylum, class, order, family, genus, species ... 9
Figure 3: Phylogenetic tree of some bacteria belonging to the phylum Bacteroidetes (according to NCBI). Phylum, class, order, family, genus ... 9
Figure 4: Phylogenetic tree of some bacteria belonging to the phylum Actinobacteria (according to NCBI). Phylum, class, order, family, genus ... 10
Figure 5: Phylogenetic tree of some bacteria belonging to the phylum Verrucomicrobia (according to NCBI). Phylum, class, order, family, genus, species ... 10
Figure 6: Flow chart of the study selection ... 23
Figure 7: Some of the gut bacteria prior to and following the fermentable carbohydrate restriction (illustrative) ... 32
LIST OF TABLES
Table 1: The Bristol Stool Scale10 ... 4Table 2: Mechanisms of some of the members in the colonic microbiota53,60–62 ... 14
Table 3: Overview over the changes of the gut microbiota in patients with IBS ... 16
Table 4: The characteristics of the four included studies ... 24
Table 5: Impact of a diet low in FODMAPs on the gut microbiota composition in IBS patients (study 1-‐4) ... 31
Table 6: A taxonomic overview over the change in the gut microbiota after dietary interventions in the four included studies ... 33
1 INTRODUCTION
1.1 IRRITABLE BOWEL SYNDROME
1.1.1 Definition, diagnoses and classification
In 2012, the World Gastrointestinal Organization (WGO) practice guideline defined irritable bowel syndrome (IBS) as “a functional bowel disorder in which abdominal pain or discomfort is associated with defecation or a change in bowel habit. Bloating, distention and disordered defecation are commonly associated features” 1. IBS is the most common functional gastrointestinal disorder (FGID), and it is a complex disorder that mostly affects the large intestine, but also, in part, the small intestine. Other symptoms include flatulence, bloating, a feeling of incomplete evacuation and mucus. This is in addition to psychiatric comorbidities, which is commonly seen among IBS patients, particularly anxiety and stress 2–4. In addition, some patients may have increased fatigue, as well as limitations in their physical capabilities 5. This may lead to increased absence from work and more frequent consultations with a physician compared to healthy individuals. In fact, IBS is the most commonly diagnosed disorder by gastroenterologists 6,7. All these taken together might give IBS patients an impaired quality of life.
Gastroenterologists diagnose IBS patients using the Rome III criteria, often referred to as the current “gold standard”. It is a symptom-based diagnostic tool, since there is currently no clear diagnostic marker for IBS 8. Diagnosing IBS by the Rome III criteria requires the presence of recurrent abdominal pain, in addition to one or a combination of other symptoms, including altered stool frequency, relief of pain following defecation, and/or altered stool form or apperance. These symptoms have to occur >3 days per month during the previous three months.
In addition, the symptoms must have been recurring for more than 6 months prior to the diagnosis 9.
IBS can be divided into subtypes using the Bristol Stool Scale (Table 1); predominant constipation IBS (IBS-C), predominant diarrhea IBS (IBS-D), or a mix between diarrhea and constipation (IBS-M). There is also an un-subtyped IBS (IBS-U), where the patients do not have diarrhea or constipation. IBS-C is determined if >25% of stools correspond to score 1 or 2 (table 1), IBS-D is determined if >25% of stools correspond to score 6 or 7, and IBS-M is
determined if 25% of stools correspond to score 1 or 2 and score 6 or 7 (Table 1). If the patient has an abnormal stool consistency that does not meet the criteria of the other subtypes, the individual will be classified as IBS-U 10. However, patients may move from one classification to another over time 1,11.
Table 1: The Bristol Stool Scale10. Transit time is slow at score 1 but increases with higher score
Score Description
1 Separate hard lumps, like nuts
2 Sausage-‐shaped but lumpy
3 Like a sausage but with cracks on the surface
4 Like a sausage or snake, smooth and soft
5 Soft blobs with clear-‐cut edges
6 Fluffy pieces with ragged edges, a mushy stool
7 Watery, no solid pieces, entirely liquid
The Rome Criteria was introduced in late 1980s by The Rome Foundation group to classify and diagnose FGIDs. In 2000, the Rome Criteria was updated to the Rome Criteria II, and further to the Rome Criteria III due to increased interest in FGIDs by gastroenterologists, psychologists and the public. The Rome III Criteria was also regarded as a vital tool for researchers to gain a better understanding of FGID, including IBS 12.
The Rome III criteria relies on the organs where the symptoms are most likely to be produced, and fall in order from the esophagus to the anus. The FGIDs are classified in six major domains for adults; bowel (category C) contains sub-categories such as functional bowel disorders that include IBS (C1), functional bloating (C2), functional constipations (C3) and functional diarrhea (C4). Symptoms like pain and change in bowel habit distinguish IBS from other GI- disorders 12.
1.1.2 Prevalence of IBS
Figure 1: Worldwide prevalence of IBS 13
The prevalence of IBS (Figure 1) varies worldwide from 1.1-45%. The pooled global prevalence rate are 11,2%, and are based on population studies from various countries worldwide 13. However, the prevalence is unevenly distributed across continents and regions of continents. Prevalence rates are for instance 5-10% in most of the European countries, USA (including Alaska), South-Africa and Australia, and slightly higher in Russia, Canada and Brazil (10-14%). The lowest prevalence rates are registered in India, China, Iran and France (<5%). Most of the population data of IBS patients in African and Asian countries, in contrast, are unavailable (N/A), and this might be due to, for instance: poor health care systems or cultural differences 13. For example, it is no general global definition of the IBS symptoms across countries, despite the existence of the Bristol Stool Scale. For example, the description of constipation in Asia is “a sense of incomplete evacuation”, whereas the Bristol Stool Scale describes constipation as passing hard stools, whether there is normal frequency of bowel movements or not (Table 1) 1.
Prevalence also varies between subtypes of IBS. Globally, the most commonly subtype was IBS-D with a prevalence rate of 40% of all the reported IBS cases, and the least common
subtype was IBS-M with a prevalence rate of 23% 14. IBS-M and IBS-D dominate in Bangladesh and India, IBS-M dominates in Brazil, and IBS-C is most common in parts of Asia, North America and Western Europe 1.
Differences in prevalence within a population is also evident. In most countries higher prevalence of IBS is found in young adults (<50 years) and a decreased prevalence with advancing age. This is, however, in contrast to countries such as Japan (prevalence rate 10- 14%), India (prevalence rate <5%) and Iran (prevalence rate <5%), where the prevalence seem to increase with advancing age 1,14. Globally, the prevalence of IBS in children varies from 6- 14% 15,16.
1.1.3 Etiology of IBS
IBS was first introduced as a concept in 1950 in the Rocky Mountain Medical Journal, and it was suggested that IBS was caused by a psychosomatic or mental disorder. Until 1985, diet (in e.g. food intolerance), psychological factors, local organic disorders (in e.g. hemorrhoids) and motility disturbances were suggested to play the main roles in the pathophysiology of IBS. In addition it was suggested that luminal distention was caused by “air traps” and accumulation of gas, and not due to increased amount of intestinal gases 17–23.
In 1990 it was proposed that psychiatric conditions including mood and anxiety disorders were associated with IBS 24, as well as depression 25. Later, stress was suggested to be the cause of these psychiatric conditions 3,9,26. Stress might result in decreased gastric emptying, increased intestinal motility and increased abdominal discomfort (visceral hypersensitivity). In 1998, it was suggested that some IBS patients had increased excretion of breath hydrogen, and this was later proposed to be associated with small intestinal bacterial overgrowth (SIBO) in a subset of IBS patients 27. SIBO is characterized by a quantitative increase of both aerobic and anaerobic coliform bacteria in the small intestine and have further been proposed to contribute to some of the symptoms in a subset of IBS patients. These include abdominal pain, bloating and altered bowel function 28–31. In 2015, a study confirmed that SIBO was detected in a subset of patients with IBS, and reported increased levels of the genera Escherichia/Shigella spp., Aeromonas spp. and Klebsiella spp in the proximal small intestine of patients with irritable bowel syndrome
32.
Another hypothesis was the possible alterations of the gut microbiota, which have been observed in animals 33 and can further lead to increased permeability of intestinal epithelial cells, referred to as leaky gut. This is a state where lumen content leaks through the epithelial barrier into underlying tissue. Also metabolites from underlying tissue can leak into the lumen.
This may in turn lead to a low-grade inflammation caused by pro-inflammatory cytokines produced by the innate immune system 9. This in turn can lead to downregulation of tight junction proteins essential for the epithelial barrier 13. Heredity may also play a role in IBS. The degree of similarity seen amongst monozygotic twins (22%) and in dizygotic twins (9%) indicate that there is a genetic component. However, the similiraties seen in the twin studies might be due to environmental factors. To which extent genetics play a role in the pathophysiology of IBS is currently incompletely understood 33.
The neurotransmitter serotonin has also been proposed to play a role in the pathophysiology of IBS. Lower levels of serotonin are found in IBS patients compared to healthy subjects. Low serotonin levels are associated with certain symptoms in patients with IBS, such as reduced small intestinal motility, fibromyalgia and chronic fatigue/ME 34. Furthermore, mast cells, which is primarily associated with allergic reactions and secretion of histamine, are more abundant in IBS patients compared to healthy controls. These cells are often found in connective tissue in the intestine around enteric neurons and have been associated with abdominal pain in IBS patients 35.
Furthermore, malabsorption of bile acid (i.e.Type 2 BAM) have been proposed to contribute to diarrhea in >25% of diarrhea predominant IBS patients in Western countries 36,37. This can cause a state where the bile acid concentrations are altered in the colon; high levels of secondary bile acids can cause diarrhea and low levels can cause constipation 36,37.
Finally, one of the the strongest risk factor for IBS is acute gastroenteritis, with a 6- to 7-fold increased risk of developing IBS, referred to as post-infectious IBS (PI-IBS) 38. This is because an enteric infection by bacteria, in e.g. Campylobacter jejuni, and possibly parasites (in e.g.
Giardia lamblia), might disturb the gut microbiota. If changes in the gut microbiota persist after the infection, this can lead to PI-IBS 39. Furthermore, antibiotics are often given to people with enteric infections, and these medications can also create a dysbiosis in the gut microbiota composition. Dysbiosis may last up to two years following an antibiotic treatment, as
demonstrated in mice, which may indicate that dysbiosis can enhance the disturbed gut microbiota in patients with PI-IBS 40. An average incidence of developing PI-IBS is estimated to be 10% following an acute gastroenteritis 38.
Together, these factors might indicate that changes in the interaction between the gut microbiota and host factors (e.g. environmental factors and diet) are important in the pathophysiology of IBS. However, whether these factors are a consequence or a cause of IBS is not known.
1.2 GUT MICROBIOTA
1.2.1 Definition and function
The gastrointestinal tract (GI) extends from the oral cavity to the anus. The GI tract contains a variety of microorganisms, mainly bacteria, referred to as ´gut microbiota´. This is a complex ecosystem with an estimated 100 trillion microbial cells. The lowest numbers are found in the stomach (10/mL), and it is gradually increasing from proximal to the distal end of small intestine (102-108/mL). Finally, the colon consists of 1012 microbes/mL 41,42.
The relationship between the host and the gut microbiota is often mutualistic, which means that both host and bacteria are benefiting from each other. The gut microbiota contributes in maintaining homeostasis within the host by aiding digestion of nutrients, protecting against pathogens, regulating gut motility, and developing gut immunity, whereas, the host provides the microbiota with a nutrient-rich and protected environment 3,43. In addition the gut microbiota produce certain vitamins (K and B) and short-chain fatty acids (SCFAs) from carbohydrate metabolism, the latter important as energy for colon enterocytes 3,42–45.
1.2.2 Taxonomy
Gut microbiota belonging to the kingdom bacteria consist of 17 families, 50 genera and more than 1000 species and they exhibit different functions and mechanisms 41. Gut bacteria is divided in the taxonomic classes: phylum, class, order, family, genus and species, and some of the bacteria belonging to the phyla Firmicutes, Bacteroidetes, Actinobacteria and Verrucomicrobioa are shown in figure 2-5. The most common phyla in the gut microbiota composition are Firmicutes and Bacteroidetes 41.
Figure 2: Phylogenetic tree of some of the bacteria belonging to the phylum Firmicutes (according to NCBI). Phylum, class, order, family, genus, species
Figure 3: Phylogenetic tree of some bacteria belonging to the phylum Bacteroidetes (according to NCBI). Phylum, class, order, family, genus
Figure 4: Phylogenetic tree of some bacteria belonging to the phylum Actinobacteria (according to NCBI). Phylum, class, order, family, genus
Figure 5: Phylogenetic tree of some bacteria belonging to the phylum Verrucomicrobia (according to NCBI). Phylum, class, order, family, genus, species
To classify the gut microbiota multiple techniques are available, however it is preferable that these techniques are cheap, rapid and gives accurate identification of the gut microbiota in their normal environment. Earlier, culture-based methods were widely used, but since many gut bacteria cannot be cultivated in the lab, results from such studies does not give accurate information about the gut microbiota in their natural environment or quantity. Now, it is standard to assess gut bacteria by sequencing bacterial DNA. There are several methods for this, but in general bacteria are collected, DNA is extracted and variable regions of the gene encoding 16SRNA are sequenced. This gives both the possibility to identify bacteria on different taxonomic levels as well as quantify the relative abundance 46,47.
1.2.3 What characterizes a healthy gut microbiota?
A diverse and a stable gut microbiota is associated with health, and can be defined by “the presence of classes of microbes that enhance metabolism, resilience of infection and inflammation, resistance to cancer or autoimmunity, endocrine signaling, and brain function”
48. This is referred to as normobiosis, a term used when microbiota associated with health benefits dominates in number over potentially harmful ones. Dysbiosis, in contrast, is a term used when the ecosystem in the gut is dominated by one or more potentially harmful microorganisms, thus creating a transient or a permanent imbalance in the gut microbiota 44.
In a healthy state, the colonic microbiota are dominated by the phyla Firmicutes and Bacteroidetes, followed by the phyla Actinobacteria, Verrucomicrobia and Proteobacteria (figure 2-5) 49,50. This profile usually remains stable, but the distribution at the Order level and beyond varies. The genera Bacteroides, Bifidobacterium, Streptococcus, Enterobacteriales, Enterococcus, Clostridium, Faecalibacterium, Eubacterium, Roseburia, Lactobacillus and Ruminococcus have been considered as the predominant fecal bacteria and are associated with health and proper gastrointestinal function 48,51. In addition the species Faecalibacterium prausnitzii is considered as a key member of the colonic microbiota 50. The colonic microbiota also consists of pathogenic bacteria within the phylum Proteobacteria, including Campylobacter jejuni, Salmonella enterica, Vibrio cholera and Escherichia coli. However, the abundance of this phylum is usually low. Based on several studies, a healthy gut microbiota may be characterized by a low abundance of the phylum Proteobacteria, and a high abundance of the genera Ruminococcus spp., Bacteroides spp., Prevotella spp and Clostridium clusters (IV and XIVa) 51,52. The healthy gut microbiota that are associated with the mucosa, referred to
as ´mucosa-associated´ microbiota, are dominated among others by the genera Akkermansia and Ruminococcus 51, 53.
Large inter-individual differences and small intra-individual differences have been observed in the gut microbiota composition, indicating that a core microbial population exists 44. This core microbial population are involved in central carbohydrate metabolism, and in some cases protein metabolism, including production of SCFAs and branch-chain fatty acids (BCFAs) 48.
1.2.4 Gut microbiota and diet interaction
Gut microbiota composition is affected by diets. Consequently, the gut microbiota can adapt to various dietary challenges, and change its fermentation. This can occur in conditions such as fermentable carbohydrate restriction, where the gut microbiota may switch from carbohydrate to protein metabolism. Some examples of this are when individuals ingest high amounts of plants (e.g. wheat), which contains dietary fiber such as resistance oligosaccharides (fructo- oligosaccharides (FOS), galacto-oligosaccharides (GOS)), they may be enriched with bacteria belonging to the genera Bacteroides, Prevotella and Bifidobacterium. Conversely, individuals that ingest diets containing high amounts of animal protein and fat may be enriched with e.g.
Bacteroides spp. 48. This shows that the gut microbiota can adapt to various dietary challenges and can change its metabolism.
As dietary challenges alter the gut microbiota composition, it is anticipated that the concentrations and types of fermentation products change as well. The gut microbiota ferment dietary carbohydrates or proteins that have escaped the digestion in the upper gastrointestinal tract. This leads to the production of various metabolites, including SCFAs, BCFAs and gases (Table 5). The most common SCFAs are butyrate, acetate and propionate. These, and in particular butyrate, are rapidly absorbed, and are used as energy source for colon enterocytes as well as for peripherally tissue (in e.g. the liver). SCFAs are associated with a numerous health-promoting properties by offering resistance to infection, anti-inflammatory properties (through G-proteins) and inhibition of pathogenic invasion by reducing pH 33,48,54,55. In addition, particularly butyrate and propionate, have beneficial effects on the glucose- and energy homeostasis 56–58. However, abnormal or elevated levels of butyrate, acetate and propionate may be negative for host health in some disorders of the gut, e.g. IBS. This includes increased
contractions in distal parts of the small intestine (ileum) as well as contribution to abdominal pain 59. Table 2 gives an overview over the gut microbiota metabolism and their end-products.
The bacteria that ferment dietary carbohydrates and indigestible carbohydrates are called
“saccharolytic” bacteria, and includes members of the genera Bifidobacteria, Bacteroides, Ruminococcus, Lactobacillus, Clostridium, Roseburia, Coriobacteriaceae, Dorea, Subdoligranulum, in addition to the species F. prausnitzii, Ruminococcus bromii and Acetivibrio cellolyticus 48,60–62. Bacteria belonging to the genera Lactobacillus and Bifidobacteria are lactate-producing, probiotic bacteria and ferment carbohydrates such as lactose and oligosaccharides (fructo- and galacto-oligosaccharides), respectively, as well as ferment nutrients that have been degraded by Bacteroides spp. 63. However, some of these saccharolytic bacteria can adapt to dietary changes and alter their metabolism, for instance by degrading proteins and amino acids. This includes members of the genera Bacteroides, Clostridium, Coriobacteriaceae, Adlercreutzia and Porphyromonas 54,57. Bacteria that utilize the end-products of sugar metabolism of other gut bacteria are called “asaccharolytic bacteria”
(e.g. Bifidobacteria spp. or Propionibacterium spp., see table 2) 57.
Protein metabolism may be associated with detrimental effects in contrast to what is the case with carbohydrate metabolism. Proteins and amino acids are mostly fermented in the distal colon and this part of the colon is often depleted for carbohydrates and the pH is therefore higher, hence leading to more efficient protein fermentation 48. This leads to production of ammonia (NH3), amines, phenols, indoles, sulfides, thiols, and BCFAs (isobutyrate and isovalerate), many of these (except BCFAs), are associated with genotoxicity in the host 64,65.
Some colonic microbiota can also produce gas following fermentation of either carbohydrates, proteins or metabolites from other GI-bacteria (Table 2). This relates to members of the genera Prevotella, Collinsella, Coriobacteriaceae and Dorea, to mention a few. If the composition of the colonic microbiota is disturbed, these metabolites can give symptoms such as bloating, distention and/or abdominal pain, typical for IBS patients. Conversely, bacteria belonging to the genera Adlercreutzia and Dialister have the ability to consume gas, thus the amount of these bacteria increase as gas-producing bacteria grows 60.
Table 2: Mechanisms of some of the members in the colonic microbiota53,60–62
Colonic microbiota Ferments/utilizes/degrades End-‐products .
Short-‐chain fatty
acids/metabolites Gases
Adlercreutzia spp. Hydrogen gas , protein metabolism Eqoul (a isoflavondiol =
nonsteroidal estrogen), BCFAs
Acetivibrio cellolulyticus Cellulose Acetate (lactate +
glucose in minor amounts)
Ethanol, CO2, H2, methane Akkermansia muciniphila Mucin-‐degrader. Polyphenols, fructo-‐
oligosaccharide, polyamines Galactose, N-‐
acetylglucosamine, disaccharides and small oligosaccharides
Bifidobacteria spp. Oligosaccharides that have been released
from more complex polysaccharides by Bacteroides spp. (In e.g. Lactose). In addition to mono-‐, manno-‐ and fructo-‐
oligosaccharides
Acetate, lactate
Bacteroidales/Bacteroides
spp. Proteins, and complex sugar polymers (in
e.g. Lactose) Acetate, succinate,
propionate, formate Hydrogen gas Clostridium spp. Undigested carbohydrates + peptides
and amino acids Butyrate, BCFAs NH4+ (to
Bacteroides spp.) Coriobacteriaceae spp. Protein and glucose Acetate, format, lactate,
BCFAs Hydrogen
gas
Dorea spp. Glucose Hydrogen
gas, CO2
Dialister spp. Hydrogen gas, CO2 Acetate, propionate
Eubacterium rectale Complex glycan or simple carbohydrates
and amino acids Butyrate, BCFAs
Faecalibacterium rectale Glucose, fructose, fructo-‐
oligosaccharides, N-‐acetylglucosamine and pectin
Lactate, butyrate,
formate
Lactobacillus spp. Lactose and other carbohydrates Lactate, (ethanol,
carbon dioxide of some species under some conditions)
Ethanol, carbon dioxide
Prevotella spp. Cellulose and xylans SCFA H2S
Propionibacterium spp. Lactate, succinate Propionate, produces vitamin B12
Porphyromonas spp. Amino acids (nitrogenous substrates) BCFAs
Roseburia spp. Starch and inulin Butyrate
Ruminococcus spp. Cellulose, mucin-‐degrader Butyrate, products of
mucins
Ruminococcus bromii Resistant starch Acetate Ethanol Ruminococcus torques Mucin-‐degrader, glucose, lactose Galactose, N-‐
acetylglucosamine, disaccharides and small oligosaccharides
From glucose:
ethanol, hydrogen and CO2
Ruminococcus gnavus Mucin-‐degrader, arabinose, maltose,
xylose, glucose Galactose, N-‐
acetylglucosamine, disaccharides and small oligosaccharides
From glucose:
ethanol, hydrogen and CO2 Subdoligranulum spp. Glucose Butyrate, lactate. Minor
acetate and succinate
1.2.5 Gut microbiota and IBS
Several studies have established that there are significant differences between the gut microbiota in IBS patients compared to healthy controls 41,66. These changes have largely been characterized as dysbiosis and linked to the pathophysiology of IBS 41,44.
Most studies have demonstrated a reduced bacterial diversity in IBS patients. Also altered proportions of specific bacteria and a difference in the variation of the gut microbiota composition is seen 41. For instance the phyla Firmicutes and Proteobacteria are increased, whereas the phyla Bacteroidetes and Actinobacteria are decreased (table 3) 67–70. Furthermore, genera Bacteorides spp. 52,70, Bifidobacteria spp. 71–73 and Faecalibacterium spp. are less abundant in patients with IBS 70. Increased relative abundances have been seen of the genera Ruminococcus spp., Clostridium spp., Dorea spp., Subdoligranulum spp., Dialister spp., Clostridium cluster XIVa., Roseburia spp., Coprococcus spp. 38,52,70, Lactobacillus spp. and Veillonella spp. 59,73,74. A recent study indicates that patients with IBS might have a microbial signature. Casen and coworkers suggested that the phyla Firmicutes, Proteobacteria and Actinobacteria, in addition to the species Ruminococcus gnavus were the predominant bacteria contributing to the dysbiosis seen in patients with IBS 44. Table 3 sums up the gut microbial changes in patients with IBS.
Table 3: Overview over the changes of the gut microbiota in patients with IBS
Increased Decreased
Firmicutes Bacteroidetes
Proteobacteria Actinobacteria
Ruminococcus spp. Bacteroides spp.
Clostridium spp. Bifidobacteria spp.
Dorea spp. Faecalibacterium spp.
Subdoligranulum spp.
Clostridium cluster XIVa - Roseburia spp.
- Coproccus spp.
Lactobacillus spp.
Veillonella spp.
Dialister spp.
Patients with IBS have demonstrably altered colonic fermentation, and some studies have observed an association between abdominal symptoms and abnormal concentration of organic acids (in e.g. p-hydroxybenzoic acid (pHBA), succinate, lactate) and SCFAs. An increase in hydrogen gas production has also been observed in IBS patients. Studies on SCFA- concentration in IBS patients are inconsistent. Both increased and decreased levels are found
75, 59.
Breath tests are used widely to assess certain functions of the gut microbiota. For instance, it is used to detect hydrogen- or methane levels in expired air as a measure of gas producing bacteria following administration of carbohydrate(s) (e.g. lactulose). The test was originally designed to detect small intestinal bacterial overgrowth (SIBO). If the breath hydrogen- or methane levels are increased, this can indicate SIBO, e.g. in patients with irritable bowel syndrome 31. However, this test have been criticized as it has shown to have a high rate of false positive results 29,31.
1.3 DIETARY TREATMENT AND MANAGEMENT OF IBS
The pathophysiology of IBS is not fully understood, and a cure does not exist yet. However, evidence exist to suggest that the gut microbiota and their metabolites play a role in the pathophysiology and dietary treatment strategies have been exploited to reduce symptoms in IBS patients. Other treatment options, except dietary strategies, may be pharmacological treatments. This includes antispasmodics and antidiarrheal for diarrhea, fiber supplementation for constipation, or supportive therapy with low-dose antidepressants to normalize GI-motility.
In addition to the antibiotics rifaximin and the anti-inflammatory agent mesalamine, have shown some efficacy in reducing the symptoms in subsets of patients with IBS, particularly IBS-D patients 76.
A low-FODMAP (Fermentable Oligo-, Di-, Monosaccharides And Polyols) diet has received a lot of attention for its effectiveness in reducing symptoms in patients with IBS 77–83. Probiotics and prebiotics are also treatment options as they manipulate the gut microbiota. Probiotics are live microorganisms which normally resides in the GI-tract and might confer a health benefit to the host when it is ingested 11,45. Prebiotics are selectively fermented ingredients that induce the growth or activity of several bacteria in the gastrointestinal tract, thus contributing to health benefits of their host 84,85. Several studies have shown that some probiotics effectively alleviates the symptoms in patients with IBS, particularly abdominal pain and bloating 86–89. Further, the gut microbiota can also be manipulated with fecal transplantation 41,90. The long-term effect of the low-FODMAP diet, pro- and prebiotics, and fecal transplantation remains unclear 4,41,76.
1.3.1 Low-‐FODMAP diet
About two-thirds of all IBS patients report that their symptoms are associated with food, particularly poorly absorbed carbohydrates, including fructose, lactose, sorbitol and other sugar alcohols 11,91,92, but foods containing high amounts of fat, biogenic amines or lectins (proteins that binds carbohydrates) might also contribute to symptoms in IBS patients. The same has been reported concerning foods containing preservatives (e.g. benzoic acid or sulfite), spicy foods (onion, garlic), as well as food that trigger histamine release 91. However, the low- FODMAP diet have been suggested as a treatment strategy for reducing the symptoms that occur in IBS patients following ingestion of poorly absorbed fermentable carbohydrates.
The low-FODMAP diet is the first reported diet to be effective in alleviating GI-symptoms in the majority of IBS patients 93. As the name implies the low-FODMAP diet is low in dietary carbohydrates that are poorly absorbed in the small intestine, including lactose, polyols, fructans and galacto-oligosaccharides. These are osmotically active due to their small size, and are rapidly fermented by the gut microbiota that reside in the colon, thus leading to increased gas production 94–96. Increased gas production is not necessarily painful to healthy individuals, but it can be painful to IBS patients, and this can be explained by more sensitive intestines (hypersensitivity) likely due to the activation of cells around enteric neurons in the intestine.
Furthermore, the malabsorption may be explained by the absence or reduced concentration of digestive enzymes in the small intestine 7, which leads to luminal distension due to fermentation in the colon, and accounts for symptoms in patients with IBS 11. Some FODMAPs are also prebiotic (e.g. oligosaccharides) and it has been suggested that a reduced production of some SCFAs, due to reduced intake of prebiotics, may reduce symptoms in IBS patients 52.
As early as in the 1980s and 1990s there was evidence to suggest that some short-chain carbohydrates, particularly lactose, sorbitol and fructose, were poorly absorbed, and played a role in the induction of IBS symptoms. This was observed in studies on dietary restriction that caused fewer GI-symptoms 77–79,83. Further examination suggested that fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and multiple sugar alcohols (e.g. mannitol), also played a role in the induction of GI-symptoms, due to the incomplete absorption 97.
In 2006, the first research trial on the role of a restricted FODMAP diet was conducted in IBS patients. The focus was to restrict fructose/fructans diet. The initial study found that 74% of all IBS patients following this diet showed decreased symptoms 81. This was confirmed by Shepherd et al. (2008), who concluded that a diet low in fructose and/or fructans may reduce GI-symptoms in IBS patients with fructose-malabsorption 80. Subsequently, Staudacher et al.
(2011) confirmed these findings in a controlled study in IBS patients, where a low-FODMAP diet was compared with a standard UK diet. Eighty six percent of the participants with IBS reported gastrointestinal symptomatic improvement following the low-FODMAP diet, compared to the UK diet 82.
Furthermore, high-quality evidence from prospective studies and randomized controlled trials supports the efficacy of a low-FODMAP diet in alleviating GI-symptoms in IBS patients,
including pain, bloating and diarrhea 98. Seventy five percent of IBS patients have reported an improvement in symptoms after following a diet low in FODMAPs 63.
The low-FODMAP-diet consists of two phases, the elimination phase and the reintroduction phase. The first phase is to consider the patient´s degree of benefit, and reduce symptoms. This is often achieved through an elimination phase, generally lasting 6-8 weeks. However, the length of this period is individual and might last for a longer or shorter period. It is recommended to be guided by a dietitian and the purpose is to be symptom-free prior to the onset of the second phase; the inclusion phase 93.
During the elimination phase, patients with IBS must avoid multiple food items containing high FODMAPs, some examples are shown in Box 1. During the reintroduction phase (second phase), the aim is to reintroduce food items to determine which food items that create symptoms. Reintroduced food items are determined on individual basis, but wheat, fiber or onion-containing foods are the most common foods to reintroduce 93.
Box 1 High FODMAPs to avoid and some food items they are presented in
Oligosaccharides (FOS, GOS) Wheat, rye, garlic, onions, legumes
Lactose Some dairy products
Fructose (particularly in excess of glucose) Pears, apples, honey, high-fructose corn syrup
Polyols (sugar alcohols) Pears, apples, artificially sweetened gums, confectionary
It is vital to consult a dietician during both phases of the diet to tailor a nutrition plan and to determine which type and what amounts of FODMAPs are tolerated. The aim is to prevent nutrient deficiency, without having GI-symptoms 93.
2 AIM
Diets low in FODMAPs, have been used widely to aid IBS patients to reduce symptoms such as bloating/distension, abdominal pain and diarrhea/constipation. However, such diets reduce the intake of prebiotics by up to 50%, hence reduce the amount of total carbohydrates available for colonic fermentation. Evidence suggest that this diet alter the colonic microbiota and further may be unfavorable to colonic health. This is particularly what this thesis aims to investigate, in addition to discuss if these effects really are unfavorable to colonic health or not. Particularly, the questions asked were:
• What changes occur in the gut microbiota composition in IBS patients following a dietary challenge with fermentable carbohydrate restriction with emphasis on low- FODMAP diets?
• Are the potential changes in gut microbiota influencing the hosts´ health?
• Are these putative effects positive or adverse to the host, and do they constitute a risk factor for the hosts´ health?
3 METHODS
3.1 Study selection
Original articles were primarily identified through selective searches using PubMed. Cochrane library were also used; however, this did not give any different results. The first search was performed using PubMed in January 2016, while an updated search was performed on April 29th 2016. Search terms used are shown in Box 2, briefly “IBS” or irritable bowel syndrome or abdominal pain”, “microbiota or microbiome”, “nutrition or diet” and “RCT or randomized controlled trial or epidemiology or pilot”. The inclusion criteria are shown in Box 3, and included: original articles, either RCTs, epidemiology or pilot studies, that explored how a diet low in fermentable carbohydrates may affect the gut microbiota composition in both adults and pediatric patients with irritable bowel syndrome (IBS) compared with diet(s) high in FODMAPs. One pilot study on pediatric patients was included due to few studies on this issue.
The exclusion criteria, shown in Box 4, were if the IBS patients received antibiotics, prebiotics or probiotics during the trial, because these criteria most likely affect the gut microbiota. Other pharmacological, complementary and alternative treatments (e.g. acupuncture, homeopathy, herbalism) were also exclusion criteria, as were other diseases or disorders (in e.g. diabetes and inflammatory bowel disease) and other diets than a diet low in fermentable carbohydrates. In this thesis the focus was on how a diet low in fermentable carbohydrates affected the gut microbiota composition. Only studies published in English were included, and search criteria were not restricted to year of publication. Figure 6 gives an overview over the study selection.
Box 2 Search terms
IBS or irritable bowel syndrome or abdominal pain Microbiota or microbiome
Nutrition or diet
RCT or Randomized controlled trial or epidemiology or pilot
Box 3 Inclusion criteria Randomized clinical trials Epidemiology trials Pilot trials
Diagnosed with IBS (using either Rome II or Rome III)
Compared the effect of a diet low in fermentable carbohydrates on gut microbiota composition with a diet high in fermentable carbohydrates
Box 4 Exclusion criteria
Use of antibiotics, prebiotics, probiotics prior and/or during the trial Pharmacological treatment
Complementary and alternative treatment (e.g. acupuncture, homeopathy, herbalism) Other diseases or disorders (e.g. diabetes, inflammatory bowel disease)
Other diets than low in fermentable carbohydrates
The search gave 31 results (figure 6), of which 27 were excluded because they did not meet the inclusion criteria: Two studies were reviews, one study was about prebiotic-supplementation, four studies were about probiotics, 14 studies did not investigate IBS, five studies did not have a dietary intervention and one study did not investigate the gut microbiota following a diet low in fermentable carbohydrates. Thus, four of the results were of interest because they met the inclusions criteria. The characteristics of these four studies that were included is shown in table 4.
Figure 6: Flow chart of the study selection
3.2 Quality of the studies using the Jadad scale
Assessment of the quality of randomized controlled trials (RCTs) is relatively new, and it exist several scales and checklists to assess the quality of RCTs. In this thesis, the Jadad scale was used (Table 5). This scale consists of three items; randomization (max. two points), blinding (max. two points) and account of all patients (max. 1 point), and can be given maximum five points in total. The RCTs can initially be given maximum two points for the item
´randomization´, this includes one point if the word ´randomization´ is mentioned in the RCTs and one point if the randomization is suitable. In contrast, one point can be removed if the method of randomization in the RTCs is unsuitable. Further can the RCTs be given two points for the item ´blinding´ if the word ´blinding´ is mentioned in the trials, and if the method of blinding is suitable. One point can, same as for the item ´randomzation´, be removed if the method of the blinding is unsuitable. Finally, the item ´account of all patients´ in RCTs can be given maximum one point if the number of patients in the RCTs and the reason why some patients in the RCTs have been eliminated (if there is any eliminations) are stated 99.
Table 4: The characteristics of the four included studies
4 RESULTS
4.1 Quality of studies and compliance to dietary interventions
In the four articles that was found eligible, 113 participants were included. Three of the studies 63,96,100 were randomized controlled trials, while the last one was an uncontrolled intervention pilot study (Table 4) 101. The quality of the RCTs was generally good. Study 2 and 3 scored 5 of 5 on the Jadad scale, whereas Study 1 received score 3 (Table 5), mainly because it was not single- or double blinded. All the four studies recruited patients with Rome III IBS (Table 4).
Study 1, 2 and 4 assessed the dietary intake both prior to dietary interventions (baseline) and after the dietary interventions 63,96,101. Study 3 only offered dietary guidance and provided menus, but did not measure the actual intake 100. In study 1, 3, and 4 96,100,101, the compliance to the intervention diets were recorded and they found good compliance. Furthermore, study 1 assessed compliance via weekly contact with the participants on the low-FODMAP group, through phone calls or emails, in addition to collect dietary diaries. Study 1 found that the intake of total carbohydrates, starch and fermentable carbohydrates following the intervention were lower in the low-FODMAP group compared with the control group (habitual diet) 96. Study 3 also collected dietary diaries and at the end of the interventions the FODMAP content was scored in a blinded fashion using a score, made for this study, for low-FODMAP ranging from 1-6. The low-FODMAP content got a mean score of 3.5, suggesting good compliance to the diet. In addition it was observed a positive correlation between the global symptom scores and their level of FODMAP ingestion 100. Finally, study 4 reported good compliance to the diet through the assessment of nutrient intake during both the habitual diet and the low fermentable substrate diet (LFSD). They found a lower total fermentable carbohydrate intake between the habitual diet and the LFSD, but no differences in numbers of fermentable items ingested between non-responders and responders were found 101. The high-FODMAP diet (Australian diet) and the low-FODMAP diet in study 2 only differed in FODMAP-content, however the authors did not report compliance to the diet 63. It was reported no significant differences in energy, protein or fat content between the low-FODMAP and the high- FODMAP diets in neither of the studies 63,96,100,101 (besides a lower total calorie-intake in pediatric participants in study 4) 101.
4.2 Effect of fermentable carbohydrate restriction on IBS symptoms, microbial metabolites and the gut microbiota composition
4.2.1 Low-‐FODMAP diet and effect on IBS symptoms
All the four studies (study 1-4) reported a reduction in symptoms in patients with IBS following a diet low in fermentable carbohydrates. The symptoms that were reduced in all the four studies were bloating, abdominal pain, flatulence, abdominal distension and tiredness. Study 4 divided the participants in responders (>50% symptom improvement) and non-responders (no or little symptom improvement) following the LFSD. In addition this study reported that responders had a trend toward lower pain frequency than non-responders, but this was not statistically significant 101.
Study 1, 2 and 4 assessed the whole gut transit time (WGTT) 63,96,101. Study 2 and 4 did not observe any changes in the intestinal transit in IBS patients following a fermentable carbohydrate restriction 63,101. However, study 4 reported that non-responders and responders had a trend toward fewer bowel movements, probably due to lower calorie intake on the LFSD compared to habitual diet 101. Study 1, in contrast, observed lower stool frequency (p=0.008), and more stools with normal consistency (p=0.02) in the low-FODMAP group. This can indicate that this diet can normalize the stool output in pediatric IBS patients. Intriguingly, it was no difference in stool consistency or the severity of self-reported diarrhea (p=0.56) between the low-FODMAP group and the high-FODMAP group, despite the restriction of the osmotically active fermentable carbohydrates 96.
4.2.2 Gut microbial products/metabolites
Study 1, 2 and 4 measured fecal SCFA levels to compare changes before and after low- FODMAP interventions. Surprisingly neither of the studies observed any significant changes (Table 5) 63,96,101. Intriguingly, study 2 observed an elevated pH of 0,2 units (p=0.008) in feces (Table 5) despite no change in SCFA-production 63. Similarly, study 4 did not observe any changes in SCFA-production 101. The authors suggest this could be due to the rapid absorption and use of SCFAs by colonic enterocytes. They also speculate that a possible change from carbohydrate- to protein metabolism, or a change of other gut bacteria could explain the elevated fecal pH associated with low-FODMAP intake. Study 2 and 4 also measured colonic branch-chain fatty acids (BCFAs) to evaluate whether a shift towards bacterial protein
